NMR resonance assignments of RNase P protein from \u3cem\u3eThermotoga maritima\u3c/em\u3e

Ribonuclase P (RNase P) is an essential metallo-endonuclease that catalyzes 5′ precursor-tRNA (ptRNA) processing and exists as an RNA-based enzyme in bacteria, archaea, and eukaryotes. In bacteria, a large catalytic RNA and a small protein component assemble to recognize and accurately cleave ptRNA and tRNA-like molecular scaffolds. Substrate recognition of ptRNA by bacterial RNase P requires RNA-RNA shape complementarity, intermolecular base pairing, and a dynamic protein-ptRNA binding interface. To gain insight into the binding specificity and dynamics of the bacterial protein-ptRNA interface, we report the backbone and side chain 1H, 13C, and 15N resonance assignments of the hyperthermophilic Thermatoga maritima RNase P protein in solution at 318 K. Our data confirm the formation of a stable RNA recognition motif (RRM) with intrinsic heterogeneity at both the N- and C-terminus of the protein, consistent with available structural information. Comprehensive resonance assignments of the bacterial RNase P protein serve as an important first step in understanding how coupled RNA binding and protein-RNA conformational changes give rise to ribonucleoprotein function

Site-Specific Stabilization of DNA by a Tethered Major Groove Amine, 7‑Aminomethyl-7-deaza-2′-deoxyguanosine

A cationic 7-aminomethyl-7-deaza-2′-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d­(G¹A²­G³­A⁴­<u>X</u>⁵­C⁶­G⁷­C⁸­T⁹­C¹⁰­T¹¹C¹²)₂ (<u>X</u> = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X⁵ imino resonance remained sharp at 55 °C. Additionally, two 5′-neighboring base pairs, A⁴:T⁹ and G³:C¹⁰, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A⁴:T⁹ base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A⁴:T⁹ base pair in the unmodified duplex, which confirmed that the overall fraction of the A⁴:T⁹ base pair in the open state of the modified duplex decreased. This was also observed for the G³:C¹⁰ base pair, where α<i>K</i>_op for the G³:C¹⁰ base pair in the modified duplex was 3.0 × 10⁶ versus 4.1 × 10⁶ for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X⁵:C⁸ and C⁶:G⁷ base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation

Observation of Two Modes of Inhibition of Human Microsomal Prostaglandin E Synthase 1 by the Cyclopentenone 15-Deoxy-Δ^12,14-prostaglandin J₂

Microsomal prostaglandin E synthase 1 (MPGES1) is an enzyme that produces the pro-inflammatory molecule prostaglandin E₂ (PGE₂). Effective inhibitors of MPGES1 are of considerable pharmacological interest for the selective control of pain, fever, and inflammation. The isoprostane, 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), a naturally occurring degradation product of prostaglandin D₂, is known to have anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ₂ can inhibit MPGES1 by covalent modification of residue C59 and by noncovalent inhibition through binding at the substrate (PGH₂) binding site. The mechanism of inhibition is dissected by analysis of the native enzyme and the MPGES1 C59A mutant in the presence of glutathione (GSH) and glutathione sulfonate. The location of inhibitor adduction and noncovalent binding was determined by triple mass spectrometry sequencing and with backbone amide H/D exchange mass spectrometry. The kinetics, regiochemistry, and stereochemistry of the spontaneous reaction of GSH with 15d-PGJ₂ were determined. The question of whether the anti-inflammatory properties of 15d-PGJ₂ are due to inhibition of MPGES1 is discussed

Structural Perturbations Induced by the α-Anomer of the Aflatoxin B₁ Formamidopyrimidine Adduct in Duplex and Single-Strand DNA

The guanine N7 adduct of aflatoxin B₁ <i>exo</i>-8,9-epoxide hydrolyzes to form the formamidopyrimidine (AFB-FAPY) adduct, which interconverts between α and β anomers. The β anomer is highly mutagenic in <i>Escherichia coli</i>, producing G → T transversions; it thermally stabilizes the DNA duplex. The AFB-α-FAPY adduct blocks replication; it destabilizes the DNA duplex. Herein, the structure of the AFB-α-FAPY adduct has been elucidated in 5′-d(C¹T²A³T⁴<u>X</u>⁵A⁶T⁷T⁸C⁹A¹⁰)-3′·5′-d(T¹¹G¹²A¹³A¹⁴T¹⁵C¹⁶A¹⁷T¹⁸A¹⁹G²⁰)-3′ (X = AFB-α-FAPY) using molecular dynamics calculations restrained by NMR-derived distances and torsion angles. The AFB moiety intercalates on the 5′ face of the pyrimidine moiety at the damaged nucleotide between base pairs T⁴·A¹⁷ and X⁵·C¹⁶, placing the FAPY C5−<i>N</i>⁵ bond in the <i>R</i>_<i>a</i> axial conformation. Large perturbations of the ε and ζ backbone torsion angles are observed, and the base stacking register of the duplex is perturbed. The deoxyribose orientation shifts to become parallel to the FAPY base and displaced toward the minor groove. Intrastrand stacking between the AFB moiety and the 5′ neighbor thymine remains, but strong interstrand stacking is not observed. A hydrogen bond between the formyl group and the exocyclic amine of the 3′-neighbor adenine stabilizes the <i>E</i> conformation of the formamide moiety. NMR studies reveal a similar 5′-intercalation of the AFB moiety for the AFB-α-FAPY adduct in the tetramer 5′-d(C¹T²<u>X</u>³A⁴)-3′, involving the <i>R</i>_<i>a</i> axial conformation of the FAPY C5−<i>N</i>⁵ bond and the <i>E</i> conformation of the formamide moiety. Since in duplex DNA the AFB moiety of the AFB-β-FAPY adduct also intercalates on the 5′ side of the pyrimidine moiety at the damaged nucleotide, we conclude that favorable 5′-stacking leads to the <i>R</i>_<i>a</i> conformational preference about the C5−<i>N</i>⁵ bond; the same conformational preference about this bond is also observed at the nucleoside and base levels. The structural distortions and the less favorable stacking interactions induced by the AFB-α-FAPY adduct explain its lower stability as compared to the AFB-β-FAPY adduct in duplex DNA. In this DNA sequence, hydrogen bonding between the formyl oxygen and the exocyclic amine of the 3′-neighboring adenine stabilizing the <i>E</i> configuration of the formamide moiety is also observed for the AFB-β-FAPY adduct, and suggests that the identity of the 3′-neighbor nucleotide modulates the stability and biological processing of AFB adducts

γ-Hydroxy-1,<i>N</i>^<i>2</i>-propano-2′-deoxyguanosine DNA Adduct Conjugates the N-Terminal Amine of the KWKK Peptide via a Carbinolamine Linkage

The γ-hydroxy-1,<i>N</i>^<i>2</i>-propano-2′-deoxyguanosine adduct (γ-OH-PdG) was introduced into 5′-d(GCTAGC<u>X</u>AGTCC)-3′·5′-d(GGACTCGCTAGC)-3′ (<u>X</u> = γ-OH-PdG). In the presence of excess peptide KWKK, ¹³C isotope-edited NMR revealed the formation of two spectroscopically distinct DNA–KWKK conjugates. These involved the reaction of the KWKK N-terminal amino group with the <i>N</i>^<i>2</i>-dG propylaldehyde tautomer of the γ-OH-PdG lesion. The guanine N1 base imino resonance at the site of conjugation was observed in isotope-edited ¹⁵N NMR experiments, suggesting that the conjugated guanine was inserted into the duplex and that the guanine imino proton was protected from exchange with water. The conjugates could be reduced in the presence of NaCNBH₃, suggesting that they existed, in part, as imine (Schiff base) linkages. However, ¹³C isotope-edited NMR failed to detect the imine linkages, suggesting that these KWKK conjugates existed predominantly as diastereomeric carbinolamines, in equilibrium with trace amounts of the imines. The structures of the diastereomeric DNA–KWKK conjugates were predicted from potential energy minimization of model structures derived from the refined structure of the fully reduced cross-link [Huang, H., Kozekov, I. D., Kozekova, A., Rizzo, C. J., McCullough, A., Lloyd, R. S., and Stone, M. P. (2010) Biochemistry, 49, 6155−6164]. Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the <i>S</i>-carbinolamine linkage was the major species due to its potential for intramolecular hydrogen bonding. These carbinolamine DNA–KWKK conjugates thermally stabilized duplex DNA. However, the DNA–KWKK conjugates were chemically reversible and dissociated when the DNA was denatured. In this 5′-CpX-3′ sequence, the DNA–KWKK conjugates slowly converted to interstrand <i>N</i>²-dG:<i>N</i>²-dG DNA cross-links and ring-opened γ-OH-PdG derivatives over a period of weeks